Escherichia coli cell-free protein synthesis (CFPS) has undergone a transformational shift from an exploratory platform used in the discovery of the genetic code to a present-day, high-yielding protein production platform[1]. This shift is fueled by the open nature of this system, allowing for rapid combination, supplementation, and optimization of the physiochemical environment for increasing protein yields and batch reaction duration[2, 3]. Now, cell-free systems are seen as a complement to in vivo protein expression and can be used as both a prototyping platform due to its simplicity, easiness, and modular design for protein expression[4-6] as well as a large-scale production platform for difficult to express proteins in vivo[7]. The transition from exploratory platform to high-yielding protein production platform has come about, at least in part, by complex strain engineering to stabilize biological substrates in the cell-free reaction mixtures[8, 9]. These genetic modifications targeted the deletion of proteins known to affect the stability of DNA[10], mRNA[8, 11], protein[12], energy[13], and amino acids[14, 15] in the cell-free reaction. In addition to strain engineering efforts, activation of multiple biological pathways[16], decreases in cost[17], and improved understanding of reaction contents makes CFPS an attractive platform for the production of new kinds of high-value proteins.
One area of great interest for the application of cell-free systems is the production of modified proteins containing non-standard amino acids. Incorporating non-standard amino acids or unnatural amino acids (NSAAs) allows for the production of proteins with novel structures and functions that are difficult or impossible to create using the 20 canonical amino acids[18, 19]. Recently, cell-free protein synthesis (CFPS) systems have been employed to increase yields of proteins bearing NSAAs[20, 21], achieve direct protein-protein conjugation[22], explore drug discovery[23], and enhance enzyme activity[24, 25].
Typically, NSAA incorporation systems use amber suppression technology to insert NSAAs into proteins, a method by which an in-frame amber (TAG) stop codon is utilized as a sense codon for assigning NSAAs[26, 27]. Amber suppression technology, however, has limited efficiency for NSAA incorporation because of the presence of release factor 1 (RF1). RF1 naturally binds the amber stop codon (TAG)[28] and prematurely terminates protein translation. Methods to counteract this competitive termination of the TAG stop codon include increasing the addition of competing tRNA[21], tagging and purifying out RF1[29], release factor engineering[30], and genomically recoding strains to remove RF1 and reassigning all occurrences to the synonymous TAA codon[31].
High-yield protein production with multiple-site incorporation of NSAAs still remains a critical challenge. As a result, optimized strains, protein production platforms, and methods for producing modified proteins containing NSAAs in high yields are needed.